The invention relates to the field of galenical formulations, which are used in particular as contrast media for the visualization of lymph nodes. The invention relates to the subject that is characterized in the claims, namely new galenical formulations that contain perfluoroalkyl-containing dye molecules and other perfluoroalkyl-containing substances.
Malignant tumors metastasize heaped in regional lymph nodes, whereby several lymph node stations can also be involved. Thus, lymph node metastases are found in about 50-69% of all patients with malignant tumors (Elke, Lymphographie [Lymphography], in: Frommhold, Stender, Thurn (eds.), Radiologische Diagnostik in Klinik und Praxis [Radiological Diagnosis in Clinical Studies and Practice], Volume IV, Thieme Verlag Stuttgart, 7th Ed., 434-496, 1984). The diagnosis of a metastatic attack of lymph nodes is of great importance with respect to the therapy and prognosis of malignant diseases. With the modern imaging methods (CT, US, and MRT), lymphogenous metastasis sites of malignant tumors are detected only inadequately, since in most cases, only the size and the shape of the lymph node can be used as a diagnostic criterion. Thus, small metastases in non-enlarged lymph nodes ( less than 2 cm) are not distinguished from lymph node hyperplasias without a malignant attack (Steinkamp et al., Sonographie und Kernspintomographie: Differentialdiagnostik von reaktiver Lymphknotenvergrbxc3x6berung und Lymphknotenmetastasen am Hals [Sonography and Nuclear Spin Tomography: Differential Diagnosis of Reactive Lymph Node Enlargement and Lymph Node Metastases on the Neck], Radiol. Diagn. 33: 158, 1992).
It would be desirable to distinguish between lymph nodes with metastatic attack and hyperplastic lymph nodes with the aid of specific contrast media. In this case, the contrast medium could be adminisitered intravasally or interstitially/intracutaneously (see above Siefert, H. M. et al., Lymphology 13, 150-157, 1980). The interstitial/intracutaneous administration has the advantage that the substance is transported directly from the scattering focus (e.g., primary tumor) by the corresponding lymph tract into the potentially related regional lymph node stations. Likewise, a high concentration of the contrast medium in the lymph nodes can be achieved with a low dose. Such markers that are to be administered interstitially were mainly used in the nuclear-medicine evaluation (with use of radioactive particles, such as, e.g., 198Au-colloid). Nuclear-medicine methods have only a very inadequate spatial resolution, however, in contrast to nuclear spin tomography with its high spatial resolution in the range of fractions of a millimeter. The direct x-ray-lymphography (injection of an oily contrast medium suspension in a prepared lymph vessel) is an invasive method that is used very rarely and that can visualize only a few lymph outflow stations. Fluorescence-labeled dextrans are also used experimentally in animal experiments to be able to observe the lymph outflow after their interstitial administration. All commonly used markers for the visualization of lymph tracts and lymph nodes after interstitial/intracutaneous administration have in common the fact that they are substances with a particulate nature (xe2x80x9cparticulates,xe2x80x9d e.g., emulsions and nanocrystal suspensions) or large polymers (see above, WO 90/14846). The previously described preparations have proven to be of value, however, based on their inadequate local and systemic compatibility as well as their small lymph passageway, which produces an inadequate diagnostic efficiency, in most cases unsuitable for indirect lymphography.
There is generally a greet need, therefore, for lymph-specific contrast media with suitable pharmaceutical and pharmacological properties. In the pharmaceutical properties, focus is placed first on the highest possible contrast medium concentration and an adequate stability. In the case of the pharmacological properties, and in addition to a diagnostically relevant lymph concentration that is as uniform as possible over several (or in the case of intravenous administration over all) lymph stations, focus is placed mainly on a quick and complete excretion of the contrast medium to avoid an unnecessary load of the entire organism. Moreover, corresponding preparations must have at their disposal an adequate local and acute compatibility.
With respect to the application in radiological practice and in addition to as simple an application as possible of corresponding preparations, the quick xe2x80x9cstart-upxe2x80x9d of the preparations is of central importance. Thus, if at all possible, it should be possible to perform imaging within a few hours after the administration of the contrast media.
Contrast media that are suitable for the visualization of lymph nodes in nuclear spin tomography are described in German Laid-Open Specification DE 196 03 033. There, perfluoroalkyl-containing metal complexes are disclosed, which are preferably used as lymphographic agents (see FIG. 1 of DE 196 03 033). Similar metal complexes that are suitable especially as blood-pool agents are described in German Laid-Open Specification DE 197 29 013.
A process for in-vivo diagnosis with use of NIR radiation is described in International Application WO 96/17628. Such diagnostic processes are at present under development. The contrast media that are described in this document are not suitable for visualizing the lymph nodes. There is therefore a need for suitable lymph-specific contrast media for these new diagnostic processes.
The object of this invention is therefore to make available new galenical formulations that are suitable as contrast media especially for the visualization of lymph nodes in the above-mentioned new diagnostic processes, and that meet the above-mentioned pharmaceutical and pharmacological requirements.
This object is achieved by the galenical formulations of this invention.
The new galenical formulations contain perfluoroalkyl-containing dye molecules, which can be used as contrast media in near-infrared diagnosis. The dyes satisfy certain photophysical and chemical requirements. They have high absorption coefficients and high fluorescence quantum yields to produce an effective signal even at the lowest tissue concentrations. The absorption maxima overlap a wide spectral range in a freely selectable manner. For detection in lower tissue layers (several centimeters under the surface), the spectral range between 600 and 900 nm is essential, while absorption wavelengths of 400 to 600 nm are sufficient for surface detection. The dyes further have a high chemical stability and photostability. When using light for fluorescence stimulation, the essential problem is the limited penetration depth of the light, which lies in the submillimeter range in the VIS but can be in the centimeter range in the NIR. With respect to the penetration depth, detection processes in surface tissue diseases, as well as soft tissues, are unproblematic. Since a considerable number of tissue changes (e.g., breast tumors, skin tumors, lymph node changes) are located on the surface, optical diagnostic processes in addition to the conventional methods are offered to perform a tissue differentiation based on different absorption and fluorescence patterns. In this case, the pronounced diffusion of light, which obtains increasing influence with growing tissue thickness, reduces both the resolution and the contrast of an optical image. Dyes that are used as so-called optical contrast media and are concentrated in the tissues that are to be detected can result in principle in an increase of the diagnostic value of optical detection processes, in which they increase the absorption of the tissue and provide an additional measurement signal with the dye-specific fluorescence, which can be detected arbitrarily often in front of a low tissue background with high sensitivity.
Preferred are dyes from the following classes:
Polymethine dyes, such as, e.g., cyanine dyes, squarilium dyes, croconium dyes, oxonol dyes, merocyanine dyes, cryptocyanine dyes;
xanthine dyes, such as, e.g., fluorescein and rhodamine and derivatives thereof;
heteroaromatic, cationic dyes, such as, e.g., oxazines, phenoxazines, thiazines, phenothiazines.
The new perfluoroalkyl-containing dye molecules are compounds of general formula I
Rfxe2x80x94Lxe2x80x94Axe2x80x83xe2x80x83(I)
in which Rf represents a straight-chain or branched perfluoroalkyl radical with 4 to 30 carbon atoms, L stands for a linker, and A stands for a dye molecule.
Linker L is a direct bond or a straight-chain or branched carbon chain with up to 20 carbon atoms, which can be substituted with one or more xe2x80x94OH, xe2x80x94COO, xe2x80x94SO3 groups and/or optionally is interrupted by one or more xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONRxe2x80x94, xe2x80x94NRCOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94NR groups or a piperazine, whereby R stands for a C1 to C10 alkyl radical, which optionally is substituted with one or more OH groups and/or is interrupted by one or more oxygen atoms.
Dye molecule A is a dye from the class of the polymethine dyes, xanthine dyes or the heteroaromatic cationic dyes. Dye molecule A is preferably a cyanine dye, squarilium dye, croconium dye, oxonol dye, merocyanine dye, cryptocyanine dye, fluorescein dye, rhodamine dye, oxazine dye, phenoxazine dye, thiazine dye or phenothiazine dye. Especially preferably dye molecule A is a molecule according to general formula II: 
in which
D stands for a fragment that corresponds to general formulas III to VI, whereby the position that is characterized with a star means the linkage with B: 
and in which B stands for a fragment that corresponds to general formulas VII to XII: 
whereby R1 and R2 represent a C1-C4 sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain C1-C50 alkyl chain, whereby the chain or parts of this chain optionally can form one or more aromatic or saturated, cyclic C5-C6 units or bicyclic C10 units, and whereby the C1-C50 alkyl chain optionally is interrupted by 0 to 15 oxygen atoms and/or by 0 to 3 carbonyl groups and/or is substituted with 0 to 5 hydroxy groups,
R3 stands for a radical xe2x80x94COOE1, xe2x80x94CONE1E2, xe2x80x94NHCOE1, xe2x80x94NHCONHE1, xe2x80x94NE1E2, xe2x80x94OE1, xe2x80x94OSO3E1, xe2x80x94SO3E1, xe2x80x94SO2NHE1, xe2x80x94E1,
whereby E1 and E2, independently of one another, represent a hydrogen atom, a C1-C4 sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain C1-C50 alkyl chain, whereby the chain or parts of this chain optionally can form one or more aromatic or saturated cyclic C5-C6 units or bicyclic C10 units, and whereby the C1-C50 alkyl chain optionally is interrupted by 0 to 15 oxygen atoms and/or by 0 to 3 carbonyl groups, and/or is substituted with 0 to 5 hydroxy groups,
and whereby R4 stands for a hydrogen atom, a fluorine, chlorine, bromine or iodine atom, or a branched or straight-chain C1-C10 alkyl chain,
b means a number 2 or 3,
and X and Y, independently of one another, mean O, S, xe2x80x94CHxe2x95x90CHxe2x80x94 or C(CH3)2.
Especially preferred perfluoroalkyl-containing dye molecules contain a perfluoroalkyl radical Rf with 6 to 12 carbon atoms, a linker L, which consists of a C1-C10 alkyl group that contains one or more oxygen atoms and/or one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONRxe2x80x94, xe2x80x94NRCO groups, in which R stands for a C1-C5 alkyl radical, which can be substituted with one or more OH groups and a cyanine dye as a dye molecule. Among the cyanine dyes, indocarbocyanine dyes, indodicarbocyanine dyes and indotricarbocyanine dyes are preferred. Especially preferred dye molecules are the following compounds: 
in which
p stands for 1, 2 or 3,
R1 and R2, independently of one another, stand for a 4-sulfobutyl, 3-sulfopropyl, 2-sulfoethyl-, 3-methyl-3-sulfopropyl, methyl, ethyl or propyl radical, and
R3 stands for hydrogen, for a radical xe2x80x94COOE1, xe2x80x94CONE1E2, xe2x80x94NHCOE1, xe2x80x94NHCONHE1, xe2x80x94NE1E2, xe2x80x94OE1, xe2x80x94OSO3E1, xe2x80x94SO3, E1, xe2x80x94SO2NHE1,
whereby E1 and E2, independently of one another, stand for a hydrogen atom or for a methyl, ethyl or a C3-C6 alkyl radical, which is interrupted by 0 to 2 oxygen atoms and/or by 0 to 1 carbonyl groups and/or is substituted with 0 to 5 hydroxy groups or stands for a poly(oxyethylene)glycol radical with 2 to 30 xe2x80x94CH2CH2O units.
The galenical formulations according to the invention further contain other perfluoroalkyl-containing compounds, e.g., perfluoroalkyl-containing metal complexes. Perfluoroalkyl-containing metal complexes and their production were already described in the German Laidxe2x80x94Open Specifications DE 196 03 033, DE 197 29 013 and WO 97/26017. These perfluoroalkyl-containing metal complexes are compounds of general formula XIV
Rfxe2x80x94Mxe2x80x83xe2x80x83(XIV)
in which Rf represents a straight-chain or branched perfluoroalkyl radical with 4 to 30 carbon atoms, and M is a molecule portion that contains 1-6 metal complexes.
Molecule M stands for, for example, a group Lxe2x80x94M1, whereby L stands for a linker, and M1 stands for a metal complex with an open-chain or cyclic chelator, which contains as central atom an atom of atomic numbers 21-29, 39, 42, 44 or 57-83. In this case, linker L is a direct bond, a methylene group, an xe2x80x94NHCO group, a group 
whereby p1 means the numbers 0 to 10, q and U, independently of one another, mean the numbers 0 or 1, and
R1 means a hydrogen atom, a methyl group, a xe2x80x94CH2xe2x80x94OH group, a xe2x80x94CH2xe2x80x94CO2H group or a C2-C15 chain, which optionally is interrupted by 1 to 3 oxygen atoms, 1 to 2 greater than CO groups or an optionally substituted aryl group and/or is substituted with 1 to 4 hydroxyl groups, 1 to 2 C1-C4 alkoxy groups, 1 to 2 carboxy groups,
or a straight-chain, branched, saturated or unsaturated C2-C30 carbon chain, which optionally contains 1 to 10 oxygen atoms, 1 to 3 xe2x80x94NR1 groups, 1 to 2 sulfur atoms, a piperazine, a xe2x80x94CONR1 group, an xe2x80x94NR1CO group, an xe2x80x94SO2 group, an xe2x80x94NR1xe2x80x94CO2 group, 1 to 2 CO groups, a group 
xe2x80x83optionally substituted aryls and/or is interrupted by these groups and/or is optionally substituted with 1 to 3 xe2x80x94OR1 groups, 1 to 2 oxo groups, 1 to 2 xe2x80x94NHxe2x80x94COR1 groups, 1 to 2 xe2x80x94CONHR1 groups, 1 to 2 (xe2x80x94CH2)pxe2x80x94CO2H groups, 1 to 2 groups xe2x80x94(CH2)pxe2x80x94(O)qxe2x80x94CH2CH2RF,
whereby
R1, RF and p and q have the above-indicated meanings, and
T means a C2-C10 chain, which optionally is interrupted by 1 to 2 oxygen atoms or 1 to 2 xe2x80x94NHCO groups.
In this case, metal complex Mxe2x80x2 stands for the following metal complexes:
a complex of general formula XV 
in which R3, Z1 and Y are independent of one another, and
R3 has the meaning of R1 or xe2x80x94(CH2)mxe2x80x94Lxe2x80x94R1, whereby m is 0, 1 or 2, and L and RF have the above-mentioned meaning,
Z1, independently of one another, mean a hydrogen atom or a metal ion equivalent of atomic numbers 21-29, 39, 42, 44 or 57-83,
Y means xe2x80x94OZ1, or 
whereby Z1, L, RF and R3 have the above-mentioned meanings,
a complex of general formula XVI 
in which R3 and Z1 have the above-mentioned meanings, and R2 has the meaning of R1,
a complex of general formula XVII 
in which Z1 has the above-mentioned meaning,
a complex of general formula XVIII 
in which Z1 has the above-mentioned meaning, and o and q stand for numbers 0 or 1, and yields the sum o+q=1,
a complex of general formula XIX 
in which Z1 has the above-mentioned meaning,
a complex of general formula XX 
in which Z1 and Y have the above-mentioned meanings,
a complex of general formula XXI 
in which R3 and Z1 have the above-mentioned meanings, and R2 has the above-mentioned meaning of R1,
a complex of general formula XXII 
in which R3 and Z1 have the above-mentioned meanings,
a complex of general formula XXIII 
in which R3 and Z1 have the above-mentioned meanings,
a complex of general formula XXIV 
in which Z1, p and q have the above-mentioned meaning, and R2 has the meaning of R1,
a complex of general formula XXV 
in which L, RF and Z1 have the above-mentioned meanings,
a complex of general formula XXVI 
in which Z1 has the above-mentioned meaning.
Such compounds and production thereof are described in German Laidxe2x80x94Open Specification DE 196 03 033 A1 and in International Patent Application WO 97/26017.
Molecule portion M according to formula XIV can further exhibit the following structure: 
whereby
q1 is a number 0, 1, 2 or 3,
K stands for a complexing agent or metal complex or salts thereof of organic and/or inorganic bases or amino acids or amino acid amides,
X is a direct bond for the perfluoroalkyl group, a phenylene group or a C1-C10 alkyl chain, which optionally contains 1-15 oxygen atoms, 1-5 sulfur atoms, 1-10 carbonyl groups, 1-10 (NR) groups, 1-2 NRSO2 groups, 1-10 CONR groups, 1 piperidine group, 1-3 SO2 groups, 1-2 phenylene groups or optionally is substituted by 1-3 radicals RF, in which R stands for a hydrogen atom, a phenyl, benzyl or a C1-C15 alkyl group, which optionally contains 1-2 NHCO groups, 1-2 CO groups, 1-5 oxygen atoms and optionally is substituted by 1-5 hydroxy, 1-5 methoxy, 1-3 carboxy, 1-3 RF radicals,
Y1 is a direct bond or a chain of general formula XXVII or XXVIII: 
xe2x80x83in which
R1a is a hydrogen atom, a phenyl group, a benzyl group or a C1-C7 alkyl group, which optionally is substituted with a carboxy group, a methoxy group or a hydroxy group,
Z1 is a direct bond, a polyglycol ether group with up to 5 glycol units or a molecule portion of general formula XXIX
xe2x80x94CH(R2a)xe2x80x94xe2x80x83xe2x80x83(XXIX)
xe2x80x83in which R2a is a C1-C7 carboxylic acid, a phenyl group, a benzyl group or a xe2x80x94(CH2)1-5xe2x80x94NHxe2x80x94K group,
xcex1 represents the binding to the nitrogen atom of the skeleton chain, xcex2 represents the binding to the complexing agent or metal complex K,
and in which variables k and m stand for natural numbers between 0 and 10, and 1 stands for 0 or 1,
and whereby
G is a CO or SO2 group.
Such compounds and the production thereof are described in German Laid Open Specification DE 197 29 013 A1.
Molecule portion A according to general formula I further can stand for a group L1xe2x80x94M2, in which L1 stands for a linker and M2 stands for a metal complex. In this case, linker L1 is a molecule portion according to general formula XXX 
in which
N represents a nitrogen atom,
A1 means a hydrogen atom, a straight-chain or branched C1-C30 alkyl group, which optionally is interrupted by 1-15 oxygen atoms and/or optionally is substituted with 1-10 hydroxy groups, 1-2 COOH groups, a phenyl group, a benzyl group and/or 1-5 xe2x80x94OR4 groups, with R4 in the meaning of a hydrogen atom or a C1-C7 alkyl radical, or B1xe2x80x94RF,
B1 means a straight-chain or branched C1-C30 alkylene group that optionally is interrupted by 1-10 oxygen atoms, 1-5 xe2x80x94NHxe2x80x94CO groups, 1-5 xe2x80x94COxe2x80x94NH groups, by a phenylene group (that is optionally substituted by a COOH group), 1-3 sulfur atoms, 1-2 xe2x80x94N(B2)xe2x80x94SO2 groups, and/or 1-2 xe2x80x94SO2xe2x80x94N(B2) groups with B2 in the meaning of A1, an NHCO group, a CONH group, an N(B2)xe2x80x94SO2 group, or an xe2x80x94SO2xe2x80x94N(B2) group and/or optionally is substituted with radical RF,
and in which a represents the binding to metal complex M2, and b represents the binding to perfluoroalkyl group RF.
In this case, metal complex M2 stands for a metal complex of general formula XXXI 
whereby R1 stands for a hydrogen atom or a metal ion equivalent of atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83,
R2 and R3 stand for a hydrogen atom, a C1-C7 alkyl group, a benzyl group, a phenyl group, xe2x80x94CH2OH or xe2x80x94CH2xe2x80x94OCH3,
U stands for radical L, whereby L and U, independently of one another, can be the same or different, however.
Such compounds and their production are described in the German patent application with file number 199 14 101.0 as well as in the examples below.
Especially preferred are metal complexes in which the central atom is a gadolinium atom (atomic number 64). Metal complexes with cyclic chelating agents are preferred compared to those with open-chain chelating agents.
Especially preferred gadolinium complexes are the gadolinium complex of 10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (for production, see WO 97/26017, Example 33),
the gadolinium complex of 10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17-heptadecafluoroheptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (for production, see DE 196 03 033, Example 2),
1,4,7-tris{1,4,7-tris(N-carboxylatomethyl)-10-(N-1-methyl-3,6-diaza-2,5,8-trioxooctane-1,8-diyl)-1,4,7,10-tetraazacyclododecane, Gd-complex}-10-(N-2H,2H,4H,4H,5H,5H-3-oxa-perfluoro-tridecanoyl)-1,4,7,10-tetraazacyclododecane (for production, see DE 197 29 013, Example 1),
1,4,7-tris{1,4,7-tris[(N-carboxylatomethyl)]-10-[N-1-methyl-3-aza-2,5-dioxopentam-1,5-diyl]-1,4,7,10-tetraazacyclododecane, Gd complex}-10-[2-(N-ethyl-N-perfluorooctylsulfonyl)-amino]-acetyl-1,4,7,10-tetraazacyclododecane (for production, see DE 197 29 013, Example 12),
the gadolinium complex of 10-[2-hydroxy-4-aza-5-oxo-7-aza-7(perfluorooctylsulfonyl)-nonyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (for production, see DE 196 03 033, Example 1),
1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(2,3-dihydroxy-propyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide}-1,4,7,10-tetraazacyclododecane, gadolinium complex (for production see examples),
1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane, gadolinium complex (for production see examples),
1,4,7-tris(carboxylatomethyl)-10-{(3-aza-4-oxo-hexan-5-ylic)-acid-[N-3,6,9,12,15-pentaoxa)-hexadexyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl]-amide]-
1,4,7,10-tetraazacyclododecane, gadolinium complex (for production, see examples),
and 1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(5-hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane, gadolinium complex (for production, see examples).
Instead of the perfluoroalkyl-containing metal complexes, other perfluoroalkyl-containing compounds can also be contained in the galenical formulations according to the invention. Such compounds are compounds of general formula XXXII
Rfxe2x80x94L2xe2x80x94G1xe2x80x83xe2x80x83(XXXII)
in which Rf represents a straight-chain or branched perfluoroalkyl radical with 4 to 30 carbon atoms, L2 stands for a linker, and G1 stands for a hydrophilic group.
Linker L2 is a direct bond, an xe2x80x94SO2 group or a straight-chain or branched carbon chain with up to 20 carbon atoms, which can be substituted with one or more xe2x80x94OH, xe2x80x94COO, xe2x80x94SO13 groups and/or optionally contains one or more xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONRxe2x80x3xe2x80x94, xe2x80x94NRxe2x80x3COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94PO4xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94NRxe2x80x3 groups, an aryl ring or a piperazine, whereby Rxe2x80x3 stands for a C1 to C20 alkyl radical, which in turn can contain one or more O-atoms and/or can be substituted with xe2x80x94COOxe2x80x94 or SO3 groups. Hydrophilic group G1 stands for a monosaccharide or disaccharide, one or more adjacent xe2x80x94COOxe2x88x92 or xe2x80x94SO3 groups, a dicarboxylic acid, an isophthalic acid, a picolinic acid, a benzenesulfonic acid, a tetrahydropyrane dicarboxylic acid, a 2,6-pyridinedicarboxylic acid, a quaternary ammonium ion, an aminopolycarboxylic acid, an aminodipolyethyleneglycolsulfonic acid, an aminopolyethylene glycol group, an SO2xe2x80x94(CH2)2xe2x80x94OH group, a polyhydroxyalkyl chain with at least two hydroxyl groups or one or more polyethylene glycol chains with at least two glycol units, whereby the polyethylene glycol chains are terminated by an xe2x80x94OH or xe2x80x94OCH3 group. Such substances are already known (see, e.g., Tetrahedron Letters, Vol. 36, No. 4, pp. 539-542, 1995). The synthesis of some of these compounds is described in detail in the examples below. Those compounds that contain a monosaccharide as hydrophilic group G1 are preferably used.
Especially preferred perfluoroalkyl-containing compounds contain a perfluoroalkyl radical Rf with 6 to 12 carbon atoms, a linker L2, which represents an xe2x80x94SO2 group or a straight-chain or branched carbon chain with up to 20 carbon atoms, which in turn contains one or more xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONRxe2x80x3xe2x80x94, NRxe2x80x3COxe2x80x94, xe2x80x94SO2 groups or a piperazine, in which Rxe2x80x3 has the above-indicated meaning, and a monosaccharide as hydrophilic group G1.
It is also possible to produce galenical formulations with three components and to use the latter as contrast media for the visualization of lymph nodes. Such formulations are described in detail in the examples below.
The substance mixtures according to the invention can be present in dissolved form in a solvent. The solvent is preferably water. The proportion of the perfluoroalkyl-containing dye molecule is between 0.1 and 10 mol % relative to the total amount of perfluoroalkyl-containing substances, preferably between 1 and 10 mol %. Preferred are mixtures that consist of perfluoroalkyl-containing dye molecules and other perfluroalkyl-containing compounds, in which the perfluoroalkyl chains have a length of 6 to 12 carbon atoms. Especially preferred are mixtures in which both the perfluoroalkyl-containing dye molecules and the other perfluoroalkyl-containing compounds have a perfluoroalkyl chain with 8 carbon atoms.
The new galenical formulations show surprising advantages in their use as contrast media. Galenical formulations that consist of perfluoroalkyl-containing dye molecules and other perfluoroalkyl-containing substances can be produced in a wide variety. These formulations are suitable for fluorescence detection and visually detectable staining of lymph nodes after interstitial or intravenous administration.
Compared to the already known contrast media for visualization of the lymph nodes, they show an improved compatibility and an almost complete excretion. The local compatibility further is also higher than in the previously known contrast media, and the new formulations simultaneously show a higher organ specificity. The concentration in the lymph nodes is higher than in the known contrast media for lymphography. If perfluoroalkyl-containing dye molecules and perfluorine-containing metal complexes are used simultaneously, it is possible to use various diagnostic processes in succession. In addition to the near-infrared diagnosis, e.g., computer or nuclear spin tomography can also be performed.
Another advantage consists in the fact that the lymph nodes assume a characteristic coloring. In addition to the NIR diagnosis, this allows an intraoperative fluorescence diagnosis of the lymph node morphology and lymph tract permeability as well as a fluorescence-supported removal of biopsies. In this case, the fluorescence-supported detection of the so-called sentinel lymph node is especially important. The sentinel lymph node is the first lymph node that drains the lymphs of a tumor area and thus also the first lymph node that is affected in a metastasis attack of lymph nodes.
The fluorescence-supported detection of these lymph nodes is carried out very much more simply than, e.g., the detection with the aid of radiopharmaceutical agents, since the detection of x-ray radiation is always more difficult than direct detection of fluorescence radiation. In addition, the stained lymph nodes are to be made visible, since the lymph nodes also can assume a characteristic coloring in the case of the corresponding dosage of the compounds according to the invention.
The dye molecules are produced in a way that is similar to methods that are known in the literature and then coupled with perfluoroalkyl derivatives. Preferred are dyes from the above-mentioned classes, which contain carboxyl groups or isothiocyanate groups. Especially preferred are those dyes that contain carboxyl groups, which after activation with use of standard reagents are reacted with amino groups that contain perfluoroalkyl derivatives with the formation of an amide group. Literature for synthesis of polymethine dyes: Bioconjugate Chem. 4, 105-111, 1993; Bioconjugate Chem. 7, 356-62, 1996; Bioconjugate Chem. 8, 751-56, 1997; Cytometry 10, 11-19, 1989 and 11, 418-30, 1990; J. Heterocycl. Chem. 33, 1871-6, 1996; J. Org. Chem. 60, 2391-5, 1995; Dyes and Pigments 17, 19-27, 1991, Dyes and Pigments 21, 227-34, 1993; J. Fluoresc. 3, 153-155, 1993; Anal. Biochem. 217, 197-204, 1994; U.S. Pat. No. 4,981,977; U.S. Pat. No. 5,688,966; U.S. Pat. No. 5,808,044; WO 97/42976; WO 97/42978; WO 98/22146; WO 98/26077; EP 0800831.
The production of the galenical formulations is carried out in that the perfluoroalkyl-containing dye molecule (component A) and the other perfluoroalkyl-containing substance (component B) are weighed and are dissolved in a suitable solvent. An especially suitable solvent is water. As already mentioned above, the proportion of the perfluoroalkyl-containing dye molecule is between 1 and 10 mol % relative to the total amount of perfluoroalkyl-containing substances. The concentration of the solution is preferably between 0.1 mmol/L and 20 mmol/L relative to the dye. This solution is then added in excess to commonly used galenical additives, such as, e.g., buffer solutions and the Ca-salt of the complexing agent. At 10 to 100xc2x0 C., the solutions are stirred vigorously. As an alternative, the solutions can be treated at 10 to 100xc2x0 C. in an ultrasound bath. Another alternative consists in that the solutions are treated with microwaves.
In substances that do not dissolve in water as individual components, it has proven advantageous to add a solubilizer, such as alcohol (e.g., methanol or ethanol) or another water-miscible solvent and then to distill off the latter slowly. The distillation can take place under vacuum. The residue is then dissolved in water, and the solution is filtered. It is also possible to dissolve each component per se separately in a solvent, then to join them and to proceed further as above.
Such produced solutions can be freeze-dried. The freeze-dried solutions can be dissolved again in water and retain, surprisingly enough, their advantageous properties. This allows a long storage time of the active ingredient.
With use as a contrast medium for the visualization of the lymph nodes, the aqueous solutions of the perfluoroalkyl-containing substances (at a concentration of between 0.1 mmol/L and 20 mmol/L relative to the dye, see above) are administered by interstitial/intracutaneous injection or intravenous injection at one or more injection sites. The administration volume relates to the species and form of administration between 0.1 ml and 30 ml, and the administered dose is preferably between 0.1 xcexcmol/kg and 10 xcexcmol/kg of body weight, relative to the dye.
After the galenic formulation is injected, light from the corresponding spectral range is irradiated for electronic stimulation of the dye that is used in the tissue. The reflected stimulation light or the fluorescence radiation that is emitted by the dye is recorded. Preferred are the methods in which the tissue irradiates over a large surface and the fluorescence radiation is triggered locally by recording with a CCD camera or the tissue areas that are to be formed are rastered with a fiber optic light guide, and the signals that are obtained are assembled by computer into a synthetic image. In this case, fluorescence can be detected and evaluated spectrally and/or by phase selection, as well as in a steady-state manner and/or in a time-resolved manner. The fluorescence images that are obtained can be produced at the same time as the white light images and are depicted above one another in a figure for data evaluation. In the case of intraoperative diagnosis, the staining can be observed visually in addition for fluorescence detection.